Ago2 protects siRNAs and microRNAs from target-directed degradation, even in the absence of 2'--methylation.

Target-directed microRNA (miRNA) degradation (TDMD), which is mediated by the protein ZSWIM8, plays a widespread role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of cells have target sites resembling those that trigger TDMD, raising the question as to whether they too might undergo such regulation by Dora, the ZSWIM8 homolog. Here, we find that some of these siRNAs are indeed sensitive to Dora when loaded into Ago1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into Ago1, these siRNAs are not detectably regulated by target-directed degradation because most molecules are loaded into Ago2, the Argonaute paralog that preferentially associates with siRNAs, and we find that siRNAs and miRNAs loaded into Ago2 are insensitive to Dora. One explanation for the protection of these small RNAs loaded into Ago2 is that these small RNAs are 2'--methylated at their 3' termini. However, 2'--methylation does not protect these RNAs from Dora-mediated target-directed degradation, which indicates that their protection is instead conferred by features of the Ago2 protein itself. Together, these observations clarify the requirements for regulation by target-directed degradation and expand our understanding of the role of 2'--methylation in small-RNA biology.